Medicinal herbal composition for treating infection

ABSTRACT

Herbal compositions derived from Kenyan plants are provided for the treatment of HIV and other infectious diseases. The herbal compositions can include the extracts of up to 14 plants, including the root of  Dovyalis abyssinica  and  Clutia robusta . Also provided are methods for extracting alkaloids and other compounds from the plants. Also provided are methods of treating a subject having an infectious disease, particularly HIV.

This application is a divisional of U.S. Ser. No. 11/352,931, filed Feb.13, 2006, which claims benefit of U.S. Provisional Application Ser. No.60/710,237, filed Aug. 22, 2005, the disclosures of each of which arehereby incorporated herein by reference into this application.

FIELD OF THE INVENTION

The present invention relates to combinations of extracts from plantsthat can be used in the treatment of infection.

BACKGROUND OF THE INVENTION

This application claims the benefit of provisional application Ser. No.60/710,237, filed Aug. 22, 2005, incorporated herein by reference in itsentirety. The following discussion of the background of the invention ismerely provided to aid the reader in understanding the invention and isnot admitted to describe or constitute prior art to the presentinvention.

Tens of millions of people world-wide are living with acquiredimmunodeficiency syndrome (AIDS), or are infected with the causativeagent, human immunodeficiency virus (HIV). In some countries insub-Saharan Africa, up to one in four adults has contracted the disease.Despite the costs and efforts spent attempting to identify new methodsof treatment, a cure for the disease has remained elusive.

Ancient societies have traditionally turned to plants for their healthneeds. Documented use of herbs to treat illnesses dates back to as earlyas 2,000 B.C. Recently, individuals have resorted to nature as remediesand medicines for the treatment of modern illnesses have been derivedfrom plants, such as for example, treatment of HIV and other infectiousdiseases.

For example, U.S. Pat. No. 5,178,865 discloses an experimental treatmentwith 56 herbs, and reports that 10 of the 56 herbs exhibit anti-HIVactivity in in vitro experiments. The 10 herbs include: Coptischineusis, Ligusticum wallichii, Ilicium eanclolatum, Isatis tinctoria,Salvia miltiorrhiza, Erycibe obtusifolia, Acanthopanax graciliatylus,Bostaurus domesticus, Inula helenium and Lonicera japonica. BothBostaurus domesticus and Lonicera japonica are further described to beable to combine with Scutellaria baicaleusis to exhibit anti-HIVactivity.

U.S. Pat. No. 5,837,257 discloses Chinese herbal medicines that exhibitin vitro antiviral activity against murine leukemia virus and HIV andfor treatment of animals and humans infected with HIV. In one of thepreferred embodiments, the Chinese herbal medicines contain hedyotis,Scutellarial barbatae herba, Lonicera flos, Prunellae spica and Solaniharba.

U.S. Pat. No. 5,989,556 discloses various herbal compositions fortreating viral infections which have shown in vitro antiviral activitiesagainst HIV. A first herbal composition contains Aeginetiae herba,Blechni rhizoma, Lespedezae herba, Polygoni cuspidati rhizoma,Forsythiae fructus, and Ligustri fructus. A second herbal compositioncontains Cirsii rhizoma and radix, Breeae radix, Baphicacanthis rhizomaand radix, Phellodendri cortex, and Bletillae tuber. A third herbalcomposition disclosed in the patent includes Aeginetiae Herba,Lonicerae, Flos, Prunellae spica and Lespedezal herba.

U.S. Pat. No. 6,696,094 discloses an herbal pharmaceutical compositionfor treating HIV/AIDS. The pharmaceutical composition contains 14ingredients, including: diffuse hedyotis, bistort rhizome, giantknotweed rhizome, Asiatic moonseed rhizome, baical skullcap root, Bovinebiliary powder, milkvetch root, barbary wolfberry fruit, sanqi, figwortroot, Chinese magnoliavine fruit, turmeric root-tuber, hawthorn fruitand Chinese angelica. Procedures are provided for the preparation of an“HIVCIDE condensate”, which can be formulated as an injectible solutionor as capsules. Results indicate that subjects injected with HIVCIDEsolution showed no symptoms of acute or chronic toxicity. Further, theHIVCIDE injection solution was effective in inhibiting pathologicalchanges in cells caused by HIV-1 in vitro. In a third experiment, theHIVCIDE injection solution was effective in reducing symptoms ofHIV-infected subjects in a treatment regime together with administrationof HIVCIDE capsules. HIV-positive subjects did not show adversereactions to HIVCIDE injection solution. It was further reported threeout of four subjects showed improvement in fatigue after treatment withHIVCIDE, and that HIV viral load studies indicated that all subjectsdemonstrated reduced HIV viral loads.

U.S. Pat. No. 6,455,078 discloses a medical herbal composition fortreating liver diseases and HIV. The composition contains 15ingredients, which includes diffuse hedyotis, bistort rhizome giantknotweed rhizome, Asiatic moonseed rhizome, baical skullcap root, bovinebiliary powder, milkvetch root, barbary wolfberry fruit, sanqi, redgingseng, figwort root, Chinese magnoliavine fruit, turmeric root-tuber,hawthorn fruit and Chinese angelica. Among the 15 ingredients, diffusehedyotis, bistort rhizome, giant knotweed rhizome, and Chinesemagnoliavine fruit are cited as being necessary to contribute to theefficacy of the pharmaceutical composition.

In U.S. Pat. No. 5,366,725, an extract from the seeds of Aeginetiaindica was prepared which exhibited excellent carcinostatic effects andpossesses interleukin-2 and interferon-gamma-inducing properties. Theextract is believed to be a macromolecular polysaccharide, which may ormay not contain Lipid A binding with protein depending on whether theextraction is conducted using butanol or phenol. The extracted substanceis soluble in water, insoluble in n-butanol, and has a molecular weightranging from 100,000 to 200,000 Daltons.

U.S. Pat. No. 5,411,733 to Hozumi, et al., discloses a variety of plantextracts for use as anti-herpes viral, anti-polioviral,anti-varicella-zoster virus, anti-measles virus, anti-cytomegalovirus(CMV), and anti-DNA and anti-RNA virus agents.

U.S. Pat. No. 5,178,865 discloses the anti-HIV activity in vitro of avariety of herbs known in China to exhibit anti-viral activity. Waterextractions of the mixtures, treatment with ethanol for precipitationand charcoal adsorption are disclosed for the preparation for theanti-HIV-active composition.

Two lignans, phyllamycin B and retrojusticiden B, have been reported tohave an inhibitory effect on HIV-1 reverse transcriptase activity. Thelignans are isolated from Phyllanthus myrtifolius Moon, a plant widelygrown in Southern China. See, for example, Chang, et al., AntiviralResearch, 27 (4), 367-374 (1995).

A mixture of aqueous extracts of Lonicera japonica flower buds,Forsythia suspensa fruits, and Scutellaria baicalensis rootbark havebeen shown to have antibacterial and antiviral properties. Subjects withsevere respiratory disease treated with the mixture responded as well asa control group on standard antibiotic therapy. See Houghton, et al.,Phytother. Res., 7(5), 384-386 (1993).

A water extract of Prunella vulgaris was reported to have anti-HIVBactivity when administered in combination with zidovudine (AZT) anddidanosine (ddI). Only a slight additive effect was observed for theadministration of an extract of Prunella vulgaris and zalcitabine (ddC).See John, et al., Abstr. Gen. Meet. Am. Sc. Microbiol., 94, 481 (1994).

Yamasaki et al. have reported the in vitro evaluation of 204 crude drugscommonly used in Japan for anti-HIV-1 activity and studies indicate thathot water extracts of Lithospermum erythrorhizon (root) and Prunellavulgaris (spike) showed strong in vitro anti HIV-1 activity with anIC₁₀₀ of 16 μg/mL. See Yamasaki, et al., Yakugaku Zasshi, 113(11),818-824 (1993).

Yao et al. have reported that water extracts of dried Prunella vulgaris(whole plant) were active in vitro for inhibiting HIV-1 replication, andshowed relatively low cytotoxicity to MT-4 cells. The extract alsodemonstrated activity in the inhibition of reverse transcriptase. Theactive factor was purified and identified as anionic with a molecularweight of approximately 10,000 Daltons. This active component may be thesame as the prunellin, as described by Tabba, et al., (1989). Thepurified extract inhibited HIV-1 replication in the lymphoid cell lineMT-4, in the monocytoid cell line U937, and in peripheral bloodmononuclear cells (PBMC) at effective concentrations of 6.3 and 12.5μg/mL, respectively. Pretreatment of uninfected cells with the extractprior to viral exposure did not prevent HIV-1 infection upon subsequentexposure to the virus. Preincubation with the purified extract decreasedHIV-1 infectiousness. The purified extract also blocked cell-to-celltransmission of HIV-1, prevented syncytium formation, and interferedwith the ability of both HIV-1 and purified gp120 to bind to CD4. PCR(polymerase chain reaction) analysis confirmed the absence of HIV-1proviral DNA in cells exposed to virus in the presence of the extract,suggesting that the purified extract antagonized HIV-1 infection ofsusceptible cells by preventing viral attachment to the CD4 receptor.See Yao, et al., Virology, 187(1), 56-62 (1992).

Tabba, et al. isolated and partially characterized prunellin, a compoundexhibiting anti-HIV properties, from aqueous extracts of Prunellavulgaris, a Chinese herb. Prunellin was identified as a carbohydrate (apartially sulfated polysaccharide) with an minimum inhibitionconcentration of 2.2 μg/mL against HIV-1 in vitro. It was identified ashaving a molecular weight of about 10,000 Dalton. See Tabba, et al.,Antiviral Research, 11, 263-273 (1989).

Antiviral agents have been isolated from Syzygium aromatica, Sapiumsebiferum (Chinese tallow tree leaves), Scutellaria baicalensis, andScutellaria rivularis. Eugeniin, (a tannin isolated from Syzygiumaromatica), and methyl gallate, (isolated from Sapium sebiferum),exhibited anti-herpes simplex virus (HSV-2) activity in vitro. Plantflavonoids, such as 5,7,4-truhydroxyflavone, extracted from the wholeherb Scutellaria rivularis, were reported to have anti-influenza virusactivity. See Hozumi, et al., U.S. Pat. No. 5,411,733; Takechi, et al.,Planta Medica, 42, 69-74 (1981); Kane, et al., Bioscience Report, 8,85-94 (1988); and Nagai, et al., Chem. Pharm Bull. 38(5), 1329-1332(1990).

Ethiopian medicinal plants known for treatment of a variety of ailmentswere screened for activity against HIV-1 and HIV-2, as reported byAsres, et al. Extracts from Bersama abyssinica root bark, Combreturnpaniculatum leaves, Dodonaea angustfolia leaves, and Ximenia Americanastem bark each displayed anti-viral activity at concentrations that werenon-toxic to MT-4 cells. Anti-viral activity of the extracts is noted tobe more effective against HIV-1 than HIV-2. See Asres, et al.,Phytother. Res., 15, 62-69 (2001).

Selected plants used in traditional Rwandan medicine for treatment ofinfections and/or rheumatoid diseases were investigated for antiviralactivity in vitro against HIV-1. See Cos, et al., Phytomedicine, 9,62-68 (2002). Of 38 plant extracts tested, extracts from the leaves ofAspilia pluriseta and Rumex bequaertii had the highest antiviralactivities.

SUMMARY OF THE INVENTION

The present invention is based upon the discovery of the uniqueantiviral properties of a herbal remedy composition prepared from avariety of plants native to Kenya. The herbal composition of the presentinvention can include plant material from between two and 14 differentplants preferably including roots of Dovyalis abyssinica (representativeseed of said line having been deposited under ATCC Accession No.PTA-6969) and Clutia robusta (representative seed of said line havingbeen deposited under ATCC Accession No. PTA-6970). For treatment ofinfectious disease, the herbal composition of plant material may beextracted to produce a liquid herbal composition or further purified toobtain alkaloid compounds from the plant material. The liquid herbalcomposition prepared from aqueous extracts from the plants hasdemonstrated effectiveness in treating HIV-positive subjects, assubjects treated with the liquid herbal composition have experiencedimprovements in CD4+ cell counts, and in some cases, complete reversalof HIV positive status.

In one aspect, the invention provides a herbal composition for treatinginfectious diseases, such as for example, HIV. The compositioncontaining plant material includes the roots of Dovyalis abyssinica andthe roots of Clutia robusta. In other embodiments of the invention, theherbal pharmaceutical composition may also include plant material, asindicated, from one or more of the following: stem bark of Prunusafricana, stem bark of Croton macrostachyus, stem bark of Acacianilotica (representative seed of said line having been deposited underATCC Accession No. PTA-7378), roots of Rhamnus prinoides, roots ofAdenia gummifera, roots of Asparagus africanus, stem bark ofAnthocleista grandiflora, whole plant of Plantago palmata(representative seed of said line having been deposited under ATCCAccession No. PTA-7377), roots of Clematis hirsuta, stem bark ofEkebergia capensis, stem bark of Bersama abyssinica, and roots ofPeriploca linearifolia.

In another aspect, the invention provides a method for preparing aliquid extract of the solid herbal composition of the invention. Theextraction of plant material can be done with hot water. In oneembodiment, hot aqueous extraction is done under basic conditions,followed by hot aqueous extraction under acidic conditions. In furtherembodiments, desired alkaloid compounds purified from the liquidextracts are provided or produced from direct chemical synthesis.

The invention further provides aqueous extracts of the herbalcompositions of the invention. Also provided are alkaloid compoundspurified from aqueous extracts and the chemical synthesis of the herbalcompositions of the invention.

In another aspect of the present invention a method for treatingHIV-positive subjects is provided. Subjects are administered aneffective amount of a herbal composition of the invention prepared fromthe aqueous extracts of Dovyalis abyssinica and Clutia robusta, alone orin combination with one or more of the following: Prunus africana,Croton macrostachyus, Acacia nilotica, Rhamnus prinoides, Adeniagummifera, Asparagus africanus, Anthocleista grandiflora, Plantagopalmata, Clematis hirsuta, Ekebergia capensis, Bersama abyssinica, andPeriploca linearifolia, in doses based on subjects' body weights. Inother embodiments the herbal composition of the invention is preparedfrom purified alkaloid compounds obtained from the aqueous extracts. Theherbal compositions are administered at least once a day. In otherembodiments, the herbal composition is administered twice or three timesdaily, based upon the health of the subject. In other embodiments, thecomposition may be administered as a beverage, capsule, tablet, powder,candy, gel, nutritional product or pharmaceutical product.

In another aspect of the present invention provides an herbalcomposition for treating subjects having infection, such as for example,HIV or AIDS. The herbal composition consists essentially of extracts ofDovyalis abyssinica and Clutia robusta, and optionally one or more ofthe following: Prunus africana, Croton macrostachyus, Acacia nilotica,Rhamnus prinoides, Adenia gummifera, Asparagus africanus, Anthocleistagrandiflora, Plantago palmata, Clematis hirsuta, Ekebergia capensis,Bersama abyssinica, and Periploca linearifolia. In one embodiment, theherbal composition of the invention is prepared from purified alkaloidcompounds obtained from aqueous extracts.

In another aspect of the present invention a method is provided fortreating subjects having infection, such as for example, HIV or AIDS.Subjects are administered an effective amount of a herbal compositionconsisting essentially of extracts of Dovyalis abyssinica and Clutiarobusta, and optionally the extract of one or more of the following:Prunus africana, Croton macrostachyus, Acacia nilotica, Rhamnusprinoides, Adenia gummifera, Asparagus africanus, Anthocleistagrandiflora, Plantago palmata, Clematis hirsuta, Ekebergia capensis,Bersama abyssinica, and Periploca linearifolia, in doses based onsubjects' body weights. In other embodiments the herbal composition ofthe invention is prepared from purified alkaloid compounds obtained fromthe aqueous extracts. The herbal compositions can be administered atleast once a day. In other embodiments, the herbal composition can beadministered twice or three times daily, based upon the health of thesubject. In other embodiments, the composition may be administered as abeverage, capsule, tablet, powder, candy, gel, nutritional product orpharmaceutical product.

As used herein, a person is considered HIV-negative if he/she has testednegative on the two-part HIV screening test (ELISA and Western blot).

As used herein, the term “therapeutically effective” or “effectiveamount” indicates that the materials or amount of material is effectiveto prevent, alleviate, or ameliorate one or more symptoms of a diseaseor medical condition, and/or to prolong the survival of the subjectbeing treated.

As used herein, “pharmaceutically acceptable” indicates that theidentified material does not have properties that would cause areasonably prudent medical practitioner to avoid administration of thematerial to a subject, taking into consideration the disease orconditions to be treated and the respective route of administration.

As used herein, the term “composition” refers to a formulation suitablefor administration to an intended animal subject for therapeuticpurposes that contains at least one pharmaceutically active compound andat least one pharmaceutically acceptable carrier or excipient.

“About” is used herein to mean in quantitative terms plus or minus 10%.

As used herein, amelioration of the symptoms of a particular disorder byadministration of a particular pharmaceutical composition refers to anylessening, whether permanent or temporary, lasting or transient that canbe attributed to or associated with administration of the composition.

As used herein, “combination” refers to any association between or amongtwo or more items. The combination can be two or more separate items,such as two compositions or two collections. It can be a mixturethereof, such as a single mixture of the two or more items, or anyvariation thereof.

As used herein, “composition” refers to any mixture. It can be asolution, a suspension, liquid, powder, a paste, aqueous, non-aqueous orany combination thereof.

As used herein, “ingredient of a pharmaceutical composition” refers toone or more materials used in the manufacture of a pharmaceuticalcomposition. Ingredient can refer to an active ingredient (an agent) orto other materials in the compositions. Ingredients can include waterand other solvents, salts, buffers, surfactants, water, non-aqueoussolvents, and flavorings.

As used herein, “pharmaceutical composition” refers a composition thatcontains an agent and one or more other ingredients that is formulatedfor administration to a subject. An agent refers to an active ingredientof a pharmaceutical composition. Typically active ingredients are activefor treatment of a disease or condition. For example, agents that can beincluded in pharmaceutical compositions include agents for treatinginfectious disease.

As used herein, “treatment” refers any manner in which the symptoms of acondition, disorder or disease or other indication, are ameliorated orotherwise beneficially altered.

As used herein, “extraction” refers to a method of separation in which asolid or solution is contacted with a liquid solvent to transfer one ormore components of the solid into the solvent.

As used herein, an extract refers to a solution containing compound(s),usually in a concentrated form, obtained by treating a solid material(such as for example, plant material) with a solvent to remove desiredcompounds or components.

As used herein, “synthesized alkaloid compounds” refers alkaloidcompounds obtained by chemical synthesis.

As used herein, “CD4+ T cell” (or “T helper cell”) refers to an immune Tcell which is involved in protecting against infectious agents includingviral, fungal and protozoal infectious agents. The CD4 molecule isexpressed on the surface of T helper cells, which also serves as theprimary target for HIV-1 and HIV-2. CD4 is the co-receptor for the Tcell receptor and recruits the tyrosine kinase Ick intracellularly. CD4+cell counts are reduced with the progression of HIV.

As used herein, “CD8+ T cell” refers to an immune T cell which hascytotoxic activity for infected cells. The CD8 molecule is expressed onthe surface of T cytoxic lymphocytes. CD8 T-lymphocyte counts increaseat the onset of HIV infection and continue to rise through theprogression of the disease.

As used herein, “CD4+/CD8+ ratio” refers to the ratio of CD4+ cells toCD8+ cells in a given sample, and is an important measure of diseaseprogression.

As used herein, “cluster of differentiation” (CD) molecules are markerson the cell surface, as recognized by specific sets of antibodies, usedto identify the cell type, stage of differentiation and activity of acell.

As used herein, the terms “HIV” and “AIDS-related virus” mean thecommonly designated HIV series (human immunodeficiency virus) andspecies thereof.

As used herein, the terms “HIV-related disease” and “AIDS-relateddisease” shall refer to any illness or syndrome, caused directly orindirectly by HIV or AIDS-related virus, including but not limited toinfections whose source is fungal, viral and/or bacterial.

As used herein, “highly active antiretroviral therapy”, or HAART, refersto treatment regimens designed to aggressively suppress viralreplication and progress of HIV disease, usually consisting of three ormore different drugs, such as for example, two nucleoside reversetranscriptase inhibitors and a protease inhibitors.

As used herein, “acute HIV infection” refers to the period of rapidviral replication immediately following exposure to HIV.

As used herein, “AIDS wasting syndrome” refers to the involuntary weightloss of 10 percent of baseline body weight plus either chronic diarrheaor chronic weakness and documented fever in the absence of a concurrentillness or condition other than HIV infection.

As used herein, “antiviral” refers to a substance or process thatdestroys a virus or suppresses replication (reproduction) of the virus.

As used herein, “viral load test” (in relation to HIV) refers to a testthat measures the quantity of HIV RNA in the blood, expressed as numberof copies per mL of blood plasma.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 shows relationships between observed clinical symptomatology andCD4+ count results.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the discovery that combinations ofextracts from plants native to Kenya can be used in the treatment ofinfection, such as for example, HIV and AIDS. Herbal compositionsprepared from combinations of the extracts of the following: the rootsof Dovyalis abyssinica and Clutia robusta, Rhamnus prinoides, Adeniagummifera, Asparagus africanus, Clematis hirsuta, and Periplocalinearifolia, the stem bark of Ekebergia capensis, Bersama abyssinica,Prunus africana, Croton macrostachyus, Acacia nilotica, and Anthocleistagrandiflora, and the whole plant of Plantago palmata have been shown tobe particularly effective in improving the health of infected subjects.Specifically, herbal compositions of the present invention areparticularly well suited for the treatment of infectious diseasesincluding HIV.

Compositions of the invention can be prepared from plant materialcollected from the Mau Forest Complex in Western Kenya. Herbalcompositions prepared from aqueous extractions and purified extracts ofplants from this region of Kenya exhibit increased potency in thetreatment of infectious diseases. The Mau Forest Complex is located at0° 30′ South, 35° 20′ East and in the Rift Valley Province, and spansfour Kenyan administrative districts: Narok, Nakuru, Bomet and Kericho.Mean annual rainfall varies from 1000 to 1500 mm with peaks in April andAugust. The rainfall pattern at the western flanks is governed by themoist monsoon winds from the Indian Ocean and dry winds from the GreatRift Valley. The western flanks of the Mau Forest Complex are influencedby the Lake Victoria macroclimatic region and are generally wetter withannual rainfall greater than 2000 mm and more evenly distributed. Meanannual temperatures for the Mau Forest Complex range from 12-16° C. Thesoil of the Mau Forest Complex is rich volcanic loam having a pH between3.8-5.8.

The vegetational pattern follows an altitudinal gradient with localtopographical ecolines. The closed canopy moist mountain forest at loweraltitudes becomes increasingly intermixed with bamboo from 2200 monwards. Between 2300 and 2500 m, pure bamboo (Arundinaria alpina)swards are found. Above 2500 m this gives way to mixed bamboo/treestands, both associated with grass clearings that usually represent asub-climax resulting from burning and cutting of bamboo. A marginal typeof mountain sclerophyll forest, wherein the plants generally have hardleaves to prevent wilting during dry conditions, occupies the highestaltitudes of the Mau complex.

Plants in the Western flank of the Mau Forest Complex have shown thehighest potency for the herbal compositions. Plants growing in theWestern flank, (which is generally a high rainfall, high altituderegion), have fewer environmental stresses. It is therefore possiblethat plants of the Western flank have more biosynthetic pathways, whichmay in turn lead to the production of a greater number of diversecompounds, which may in turn explain the greater potency of plants fromthe Western flank (as compared to other regions of the Mau ForestComplex). Alternatively, the greater potency plant extracts from theWestern flank plants may be a result of a greater variety and number ofalkaloids and other compounds in the plant extracts, such that thecombined effect is greater than the sum of their individual effects.

The East Mau Forest Complex has a drier vegetation of Cedar and Podo.Wherever these species have been extracted, colonizing species such asNeuboutonia marcrocalyx and Macaranga capensis can be found.

The compositions of the invention may be prepared using plants collectedfrom three altitude ranges of the Mau Forest Complex: 2000 m (annualrainfall of 1000 mm), 2300 m (1500 mm), 2500 m (western Mau flank,annual rainfall greater than 2000 mm) above sea level. The Westernflanks of the Mau Forest contain plants that are particularly preferredfor preparing the herbal compositions of the invention. The plants grownin the drier Eastern flank of the Mau Forest Complex also may be used.

Plant material for preparing compositions of the invention may also beobtained from plants grown in a greenhouse environment. The germinationof the seeds of particular plants may be altitude or soil dependent.Seeds for greenhouse planting may require collection from the naturaldispersal agents as they exist in the wild. Additionally, simulation ofrainfall, sunlight (an average of 12 hours per day in the Mau ForestComplex), and soil conditions of the Mau Forest Complex (i.e., richvolcanic loam having a pH between 3.8-5.8) may be required to obtainplants of similar potency.

The seeds of Dovyalis abyssinica (representative seed of said linehaving been deposited under ATCC Accession No. PTA-6969) are containedin a fleshy fruit. There are about 4 seeds enclosed by the flesh. A ripefleshy fruit can be soaked in water for about 4 days, to make itpossible to squeeze with minimum force to release the small seeds, eachbeing approximately the size of a tomato seed or slightly larger. Theseeds are then washed, dried and stored, awaiting germination under MauForest-like environmental conditions. In the wild, the fruit flesh issoaked by rain water, which results in the release of the seeds. Theseeds grow naturally under the environmental conditions of the MauForest Complex as described above.

The Clutia robusta (representative seed of said line having beendeposited under ATCC Accession No. PTA-6970) seeds are much smaller andencased in berries having a nut-like outer covering which encasesapproximately 3 to 4 seeds the size of a grain of sand. When matureseeds are exposed direct sunlight, they disperse rapidly in a processcalled explosive dispersal. This is not a problem in the wild, but ifone is interested in collecting the seeds, care and intelligence arerequired, or else all the seeds will fly away under the scatteringeffect of the hot sun.

To recover the Clutia robusta seeds, the berries should be placed in ametallic container, and covered with a material that allows sunlight toenter, such as a transparent polyethylene film surrounding a containerof appropriate wire mesh. Exposure to light will cause the shells tobreak open, releasing the seeds which can then be separated from thechaff.

The optimal time for planting the clutia and dovyallis seeds in theirnatural environment is during the long rains, typically around the monthof April. However, in the wild, the plants will generally growthroughout the year, except during the dry season, as the plants requirea considerable amount of water and light to grow.

Croton macrostachyus produces pale pea-sized capsules, on droopingspikes to 30 cm long, splitting open on the tree to release 3 shiny greyseeds, covered at one end by a soft, creamy aril, or envelope.

Prunus africana produces spherical fruit, about 10 mm in diameter and ispinkish brown in color.

The Acacia nilotica (representative seed of said line having beendeposited under ATCC Accession No. PTA-7378) plant produces straight orcurved pods measuring approximately 17×2 cm. When young, the pods aregreen and fleshy but get darker with age, and are usually velvety. Podshave a fruity odor and open on the ground to release seeds.

Ekebergia capensis produces rounded, thin skinned berries, up to 2.5 cmin diameter, on long stalks in heavy bunches, which are yellow to red incolor when mature.

The berry-like fruits of Rhamnus prinoides are approximately the size ofa pea (about 5 mm in diameter), roundish and clearly divided into threecompartments. They are fleshy and green, turning red and then purple asthey ripen.

The fruit of the Asparagus africanus is a round berry, approximately 0.5cm in diameter, green aging to orange, found most of the year. It isspread mainly by birds carrying the seeds.

The Anthocleista grandiflora produces fruits that are oval in shape,measuring approximately 3 cm×2 cm, glossy, smooth and brown when mature.Multi-seeded, large fruits are found throughout the year.

The Bersama abyssinica produces a smooth, spherical capsule, measuringapproximately 2.5 cm in diameter, golden velvety at first, losing mostof the hair and becoming brown by maturity; splitting into four valvesto reveal attractive bright red seeds, about 10 mm long, enveloped forabout their half length by a yellow, cup-shaped aril.

Adenia gummifera produces a fruit which is a stalked 3-valved capsule,leathery or fleshy, often red; seeds compressed with bony testa in afleshy aril.

Plantago palmata (representative seed of said line having been depositedunder ATCC Accession No. PTA-7377) produces a capsule-like fruit withtwo seeds per capsule.

Periploca linearifolia (representative seed of said line having beendeposited under ATCC Accession No. PTA-7375) produces black seedsmeasuring approximately 10 mm long and 2 mm wide with white woolmeasuring around 3 cm attached to the tips of the seeds. The seeds areenclosed in pods measuring about 12 cm long. Upon maturity, the podsbreak open upon exposure to sunlight. This releases the seeds, which areborne aloft by the wool as they are dispersed by wind. Alternatively,these plants may be cultivated from stem cuttings, which when laid on orplanted in the ground, grow roots and propagate new plants.

Clematis hirsuta (representative seed of said line having been depositedunder ATCC Accession No. PTA-7383) produces yellowish seeds measuringapproximately 3 mm in length and 1 mm in breadth. The seeds aresurrounded by yellowish-white wool which measures about 5 mm long. Thewool carries the seeds upon the wind, which is the dispersal agent.

HIV Testing

As noted previously, for purposes of this application, a person isconsidered HIV-negative if the subject tested negative on a two-part HIVscreening tests, consisting of an initial screening test and aconfirmatory test.

An infected individual usually goes for testing for one or more of thefollowing reasons: 1) the individual feels ill, 2) the individual'ssexual partner is ill and has tested positive, 3) the individual'ssexual partner died of AIDS; or 4) the individual suspects his/hersexual partner is sexually promiscuous.

The initial screening test is ELISA (Enzyme-Linked Immunosorbent Assay),an enzyme immunoassay (EIA) to determine the presence of HIV antibodies.The ELISA test uses artificial HIV proteins that capture antibodies tothe virus and is more than 99 percent accurate. If antibodies to HIV arepresent (positive result), the test is typically repeated. However,other antibodies can cause a false-positive result.

Generally, HIV-1 antibodies are detectable approximately 25 days afteracute infection, with nearly all infected subjects testing HIV positive12 weeks after infection. The process of developing antibodies to avirus is termed seroconversion, and individuals who becomeantibody-positive are often called seroconverters.

Two types of HIV have been identified: HIV-1 and HIV-2, of which, HIV-1is more common. HIV-1 and HIV-2 are similar in the modes of transmission(sexual contact, sharing needles, etc.) and infected individuals aregenerally subject to the same opportunistic infections. However, HIV-2appears to weaken the immune system more slowly than HIV-1.

In Kenya, individuals are generally tested for antibodies to both HIV-1and HIV-2. HIV-1 is generally more common in the Western world and HIV-2is more common in Africa. In Kenya however, most HIV-positiveindividuals have the HIV-1 infection. It is believed that 90% of theHIV-positive cases in Kenya are HIV-1, with the remaining 10% ofHIV-positive cases being the HIV-2. While rare, subjects occasionallyare HIV antibody-positive to both types of HIV (i.e. HIV-1 and HIV-2).

The second part of the HIV screening test is called the confirmatorytest. In the U.S., the most often used confirmatory test is the Westernblot, wherein an electrical field is used to separate the variouscomponents by their molecular weight prior to evaluating antibodybinding. This allows identification of antibodies to specific viralantigens, which show up as identifiable “bands” on a strip of testpaper. The Western blot test is more difficult to perform and accuratelyinterpret than the ELISA test, but it is less likely to give afalse-positive result because it can distinguish HIV antibodies fromother antibodies that may react to the ELISA. Other confirmatory testsmay be used, including the indirect fluorescent antibody assay (IFA) andthe radioimmunoprecipitation assay (RIPA).

One major drawback of antibody tests is the “window” period (i.e. thetime it takes the body to produce antibodies after infection has begun).The screening tests do not correlate to the presence or absence ofsymptoms. The standard HIV tests do not detect the virus itself, butinstead detect the antibodies that the body produces in response to thevirus. During the period before the antibodies are produced, a personmay be infected with HIV and can infect others, and still test negativeon the HIV antibody test. It is therefore important to tell subjects whotest negative to avoid engaging in high-risk behavior and to return forretesting at a later date.

The p24 antigen test can be used in diagnosing HIV early in the courseof infection. It is primarily used to screen the blood supply but insome places it is used for testing for HIV. The p24 antigen is a proteinthat is part of the HIV. Early in the infection, it is produced inexcess and can be detected in the blood serum by a commercial test. Thep24 test can detect HIV infection before the HIV antibody test can andit is recommended 2-3 weeks after a risk exposure.

Individuals that test positive for HIV are regularly administered twotests to monitor HIV levels in the blood and to determine how the virusis affecting the immune system. These tests are: (1) a viral loadmeasurement, and (2) CD4+ cell counts.

Viral load measurement (also called the HIV plasma RNA test) determineshow many HIV viral particles are present in a given amount of a person'sblood. Test results help determine the best treatment for the HIVinfection as the viral load test shows how fast the virus is multiplyingin the body. Because HIV reproduces by making copies of itself, theresults are given as copies per milliliter (mL). Viral load testing canalso reveal the presence HIV infection before antibodies can be detectedand can also accurately determine whether a baby born to an infectedmother has HIV.

CD4+ cell counts (T-lymphocyte measurements) provide an estimate of theimmunologic status of an individual and help determine the immediaterisk of opportunistic infection. The CD4+ count measures the number of acertain type of white blood cell that is most affected by HIV, and aremeasured every 3 to 4 months in individuals infected with HIV. Onaverage, an individual infected with HIV loses approximately, 30-60 CD4+cells per year, although in some subjects, CD4+ T-lymphocyte counts mayremain stable for years followed by rapid decline.

CD4(T4) or CD4+ cells are a type of T cell involved in protectingagainst infections, such as for example, viral, fungal, and protozoalinfections. Destruction of these cells is the major cause ofimmunodeficiency observed in AIDS, and decreasing CD4+ lymphocyte countsappear to be the best indicator for the potential development ofopportunistic infections. In judging the severity of HIV/AIDS cases, theCD4+ lymphocyte count is more indicative of the severity of the diseasethan gross symptomalogy, although it is also true that certain symptomsmay be associated with particular CD4+ lymphocyte levels. See, forexample, FIG. 1. Average normal adult CD4+ cell counts typically rangesfrom 500 to 1,500/2,000 cells per cubic milliliter of blood.

As CD4+ cell counts decrease below the normal adult levels duringprimary HIV infection, CD8+ or cytotoxic T-lymphocytes also increase.However, most studies indicate that an increase in CD8 count is not aprognostic indicator of disease progression. Some clinicians in the U.S.use the CD4/CD8 ratio as an indicator of disease progression, however,this ratio varies not only with the severity of the disease, but withthe ethnicity of the subject.

There are several systems for classifying and staging HIV infection. Themost commonly-used system is the CDC (Centers for Disease Control)Scheme. The CDC scheme has three classifications based upon CD4 counts.The definitions of the three CD4+ T-lymphocyte categories 1 as follow:Category 1: >500 cells/mm³ (or CD4%>28%); Category 2: 200-499 cells/mm³(or CD4% 14%-28%); and Category 3: <200 cells/mm³ (or CD4%<14%).

In addition to the CDC classification scheme, there are also 3 possiblecategories of clinical conditions, which are designated by the lettersA, B and C. Therefore, a given individual can have the following CDCclassification and clinical categorization designation: 1-A, or 1-B, or1-C, 2-A, 2-B, 2-C, 3-A, 3-B or 3-C.

An individual in category A is identified as an adolescent or adult (>13years) with documented HIV infection having one or more of the followingconditions (and lacking any of the conditions associated with categoriesB and C): asymptomatic HIV infection; persistent generalizedlymphadenopathy; and acute (primary) HIV infection with accompanyingillness or history of acute HIV infection.

An individual in category B is identified as an adolescent or adult (>13years) with documented HIV infection having one or more of the followingconditions (and lacking any of the conditions associated with categoryC) and that meet at least one of the following criteria: (a) theconditions are attributed to HIV infection or are indicative of a defectin cell-mediated immunity; or (b) the conditions are considered byphysicians to have a clinical course or to require management that iscomplicated by HIV infection. Examples of conditions in clinicalcategory B include but are not limited to: bacillary angiomatosis;candidiasis (oropharyngeal, i.e. thrush); candidiasis (vulvovaginal,persistent, frequent, or poorly responsive to therapy); cervicaldysplasia (moderate or severe/cervical carcinoma in situ);constitutional symptoms, such as fever (body temperature of 38.5° C. orgreater) or diarrhea lasting longer than 1 month; hairy leukoplakia(oral); herpes zoster (shingles), involving at least two distinctepisodes or more than one dermatome; idiopathic thrombocytopenicpurpura; listeriosis; pelvic inflammatory disease (particularly ifcomplicated by tubo-ovarian abscess); and (11) peripheral neuropathy.For classification purposes, Category B conditions take precedence overCategory A conditions. For example, an individual previously treated fororal or persistent vaginal candidiasis (but not exhibiting a Category Cdisease or condition) who is now asymptomatic, should be classified inCategory B.

An individual in category C is identified as an adolescent or adult (>13years) with documented HIV infection having one or more of the followingconditions Category C conditions include the following: candidiasis ofbronchi, trachea, or lungs; candidiasis (esophageal); invasive cervicalcancer; coccidioidomycosis (disseminated or extrapulmonary);cryptococcosis (extrapulmonary); cryptosporidiosis (chronic intestinal,greater than 1 month's duration); cytomegalovirus disease (other thanliver, spleen, or nodes); cytomegalovirus retinitis (with loss ofvision); encephalopathy (HIV-related); herpes simplex: chronic ulcer(s)(greater than 1 month's duration), or bronchitis, pneumonitis, oresophagitis; histoplasmosis (disseminated or extrapulmonary);isosporiasis (chronic intestinal, greater than 1 month's duration);Kaposi's sarcoma; lymphoma (Burkitt's, or equivalent term), lymphoma,(immunoblastic, or equivalent term); Lymphoma (primary, of brain);mycobacterium avium complex or M. kansasii, disseminated orextrapulmonary; mycobacterium tuberculosis, (any site, pulmonary orextrapulmonary); mycobacterium, (other species or unidentified species,disseminated or extrapulmonary); pneumocystis carinii pneumonia;pneumonia (recurrent); progressive multifocal leukoencephalopathy;Salmonella septicemia (recurrent); toxoplasmosis of brain; and wastingsyndrome due to HIV. For classification purposes, once a Category Ccondition has occurred, the individual will remain in Category C.

One method of treatment for HIV-positive individuals is the highlyactive antiretroviral therapy (HAART) regimen. HAART is a therapeutictreatment regime consisting of the combination of anti-HIV drugs, thatis prescribed to HIV-positive individuals even before they developsymptoms of AIDS. The therapy usually includes one nucleoside analog,one protease inhibitor and either a second nucleoside analog or anon-nucleoside reverse transcription inhibitor (NNRTI). Frequently, theHAART regime is toxic to the individual, resulting in adverse sideeffects. For example, HAART can be toxic to blood because it almostalways includes one or two nucleoside analogs, like AZT that arenotorious for their toxicity to red and white blood cells and blood cellproduction. Various forms of anemia are very common and sometimes areirreversible. However, it is extremely rare for a subject on the HAARTregimen reverse his/her HIV status in Kenya.

Examples of drugs administered for the HAART treatment regime include:azidovudine (AZT), didanosine (dideoxyinosine, ddI), zalcitabine(dideoxycytosine, ddC), lamivudine (epivir, 3TC), nevirapine (Viramune),abacavir (Ziagen), stavudine (Zerit, d4T), tenofovir (Viread), efavirenz(Sustiva), amprenavir (Agenerase), lopinavir (Kaletra), nefinavir(Viracept), saquinavir (Invirase), ritonavir (Norvir), indinavir(Crixivan), and delavirdine (Rescriptor).

Method for Extracting Alkaloid Compounds and Preparing HerbalComposition

The compositions of the invention are prepared using roots of Dovyalisabyssinica and Clutia robusta, and optionally one or more of thefollowing: the stem bark of Prunus africana, stem bark of Crotonmacrostachyus, stem bark of Acacia nilotica, roots of Rhamnus prinoides,roots of Adenia gummifera, roots of Asparagus africanus, stem bark ofAnthocleista grandiflora, whole plant of Plantago palmata, roots ofClematis hirsuta, stem bark of Ekebergia capensis, stem bark of Bersamaabyssinica, and roots of Periploca linearifolia. Preferably, theingredients collected are fresh, although dried samples may also beused. The ingredients are combined and chopped into small pieces anddried. Preferably, the dried ingredients are ground into a fine powderafter drying. Alternatively, each ingredient may be processedindividually and combined at a later stage. Preferably, if combined forthe extraction process, the ingredients are combined in equal weightratios. Optionally, Dovyalis abyssinica, Clutia robusta, Prunusafricana, Croton macrostachyus, Acacia nilotica, Rhamnus prunioides,Adenia gummifera, Asparagus africanus, Anthocleista grandiflora,Plantago palmata, Clematis hirsuta, Ekebergia capensis, Bersamaabyssinica and Periploca linearifolia can be present in a weight ratioof 2:2:2:2:2:2:1:2:2:1:2:2:2:2.

The herbal plant material mixture may be extracted with a non-polarsolvent to remove fats from the chopped herbal ingredients. Preferably,approximately 20% by volume non-polar solvent is added to the herbalingredient mixture. Non-polar solvents are generally organic solventshaving a dielectric constant less than 20. Non-polar solvents that maybe used include, but are not limited to: alkanes, 1,4-dioxane, carbontetrachloride, chlorofomm, methylene chloride, benzene, ethers, ethylacetate, tetrahydrofuran, acetic acid, butanol, chlorobenzene,cycloalkanes, xylene, and the like. Preferred non-polar solvents arexylene and ether.

The non-polar solvent is decanted and discarded. The defatted herbalsolids, are then allowed to dry. Sufficient base is added to thedefatted herbal material to achieve a pH of approximately 8. Theconcentration of the base added can be adjusted to provide sufficientliquid volume to cover the defatted herbal solid mixture. Any suitablebase may be used, with preferred bases including NaOH, KOH, Ca(OH)₂,Mg(OH)₂, NH₄OH, and the like. The base extract is then heated for 2-4hours. Preferably, the ingredients are slowly simmered under refluxconditions, although the same effect can be achieved by simmering themixture in a covered pot.

Acid is added to the base extract to achieve a pH of approximately 3.Preferably the acid is HCl, although other acids, including but notlimited to, HBr, HNO₃, H₂SO₄, H₃PO₄, or any other acid suitable forachieving a pH of approximately 3 may be used as well. The concentrationof the acid can be adjusted as necessary to provide sufficient volume tothe mixture. The acidified solution is then boiled for approximately 2-4hours under the same conditions employed for the heating of the basicsolution. After heating, the mixture is cooled, and the aqueous layer isseparated from the mixture, such as for example, by decanting the liquidfrom the remaining solids. Acid is then added to the remaining residuesufficient to achieve a pH of approximately 3, and the mixture is thenreheated for approximately 2-4 hours under the same conditionspreviously employed. The aqueous layer is separated from the ingredientsand the two acidified layers are combined. If necessary, additional acidextractions may be performed.

The acidic filtrate is extracted several times with a non-polar solventuntil little or no emulsion forms. Preferable non-polar solvents areether and xylene. Base is added to the aqueous layer to precipitate thealkaloid compounds. Preferably, base is added to achieve a pH ofapproximately 9. The precipitate is separated from the aqueous solution,neutralized and dried.

The precipitate is preferably collected in either crystalline or powderform, and may administered to an subject as a beverage, capsule, tablet,powder, candy, gel, nutritional product or pharmaceutical product.

The precipitate can be further purified as desired to isolate individualalkaloid compounds by any known chromatographic means.

It is understood that at any point during the process of extracting thealkaloid compounds from the herbal ingredients that the aqueous solutioncan be concentrated and stored for later use without the need forprecipitation of the compounds from solution.

Alternatively, the alkaloid compounds for use in the present inventioncan be synthesized by known methods once the chemical structure has beendetermined. Isolated compounds can be analyzed by chemical analysis,mass spectroscopy, infrared spectroscopy, X-ray diffraction, NMR(including ¹H NMR, ¹³C NMR, COSY, NOSEY, and the like), and other knownanalytical techniques to obtain the chemical structures. For example,chemical structures for four extracts obtained from Dovyalis abyssinicahave been previously determined. (See, for example,http://www.dfuni.dk/uploads/media/Naturstofgruppen_BonnieRasmussen.pdf).

The invention will now be described in greater detail by reference tothe following non-limiting examples.

EXAMPLES

Determination of Bioactivity of Plant Extracts

The efficacy of the individual plant extracts were tested againstEscherichia coli and Staphylococcus aureus. Plant extracts were obtainedas described above. Solutions containing 100 ppm (parts per million) ofeach plant extract were prepared for use in the anti-bacterial assay.

Preparation of Bacterial Culture of Escherichia coli and Staphylococcusaureus.

Standard cultures of E. coli (representing gram-negative strains ofbacteria) and Staphylococcus aureus (representing gram-positivebacteria) were obtained from Moi University Teaching and ReferralHospital. Assays were conducted at the Moi University Department ofBotany.

Bioassay Procedure (Diffusion Method)

Nutrient agar was used as growth medium for both bacteria samples. Theagar was sterilized in an autoclave at 120° C., cooled and poured intosterile Petri dishes and allowed to set. Sterile conditions wereachieved and maintained by exposing the area to a UV lamp during samplepreparation and the assay the procedure.

The cooled agar medium was streaked on the surface with each bacteriaculture. Wells were dug in the middle of the medium, using a cork borer,where the prepared plant extract was deposited. A control experiment wasalso performed, using plain sterile water in place of the plantextracts.

Cultures were incubated for 12 hours, after which zones of inhibition ofbacterial growth were determined and measured. Bacteria-growthinhibition was expressed in diameters (mm), and was determined bymeasuring the distance from edge of the well to area where the bacteriabegin to show growth. Generally, the larger inhibition diameterindicates greater potency of the particular extract against thebacteria.

Of the 23 plants were screened in this assay, 14 of the plants hadbacteria growth inhibition diameters greater than 8 mm, which waspreviously determined to be the minimum activity required for adoptionof the extract for the herbal remedy. The anti-bacterial activities ofthe plants were compared with standard antibiotics. Of the 14 plantshaving inhibition diameters greater than 8 mm, Dovyalis abyssinica andClutia robusta demonstrated the greatest anti-bacterial activity.Results for plant extracts exhibiting inhibition diameters greater than8 mm are provided in the Table 1.

TABLE 1 Zones of Inhibition Expressed as Inhibition Diameter (mm) PlantName E. coli S. aureus 1. Dovyalis abyssinica 17.2 16.6 2. Clutiarobusta 16.7 15.8 3. Prunus Africana 14.7 14.6 4. Croton macrostachyus14.7 14.4 5. Acacia nilotica 13.6 13.2 6. Ekebergia capensis 12.8 13.07. Clematis hirsuta 11.9 12.8 8. Adenia gummifera 11.7 12.8 9. Asparagusafricanus 11.3 11.2 10. Plantago palmata 11.0 11.0 11. Rhamnus prinoides10.9 10.8 12. Periploca linearifolia 10.9 10.6 13. Bersama abyssinica10.5 10.3 14. Anthocleista grandiflora 10.0 9.7Administration of the Herbal Composition

The plant extract precipitates are preferably purified and collected ineither crystalline or powder form. The precipitates can administered toa subject as a beverage, capsule, tablet, powder, candy, gel,nutritional product or pharmaceutical product. Preferably, between 0.1and 25 grams of alkaloids are administered per day to an infectedsubject. The herbal composition is preferably administered as a beveragewherein approximately 1 tbsp of powdered extract is dissolved inapproximately 250 mL of hot water, and drunk. Dosing is either twicedaily at 12 hour intervals, or three times daily at eight hour intervals(depending on the level of infection of the test subject), and ispreferably administered with a meal.

Subjects in the current trials were screened at the Walter Reed Hospitalof the U.S. Army in Kericho, Kenya, the Moi University Hospital inEldoret, and at various Voluntary Counseling and Testing (VCT) Centersscattered throughout the country.

Subjects' CD4 and CD8 counts were measured using a FACSCount™ systemfollowing procedures provided in the FACSCount White Paper (July 1994).HIV-1 and HIV-2 antibodies were detected using a bioMerieux Vironostika®HIV Uni-Form II Ag/Ab ELISA system.

All subjects administered the herbal composition were HIV-positiveadults. Prior to administration of the herbal composition, an initialCD4 count for each subject was determined, followed by an assessment ofthe level of opportunistic infections. Those with fewer opportunisticinfections were administered the herbal composition twice daily aftermeals, at twelve hour intervals. Those with more opportunisticinfections were administered the herbal composition three times daily,at 8 hours intervals. Each subject was given one week's dosage duringeach visit to the clinic. This was done to make it possible to monitorcompliance, and to avoid the possibility of subjects sharing the drugwith others.

Example 1

Initial studies for the treatment of HIV positive subjects with herbalremedy were conducted by treating four HIV positive subjects with twodifferent herbal remedies. Two subjects were administered a herbalcomposition which included the extract of Dovyalis abyssinica, while theother two subjects were administered a herbal remedy which included theextract of Clutia robusta. The subjects were each treated for a periodof three months. The CD4 counts of both sets of subjects (i.e., thoseadministered either Dovyalis abyssinica or Clutia robusta) increased byapproximately 10 per month of treatment.

Example 2

In another study, three subjects were administered a herbal compositionprepared with a 1:1 ratio by weight mixture of Dovyalis abyssinica andClutia robusta for a period of approximately three months. The CD4counts of the subjects treated with the mixture increased byapproximately 30 per month.

Example 3

In yet another experiment, 21 subjects were administered a herbalcomposition containing extracts of Dovyalis abyssinica, Clutia robusta,Prunus Africana, Croton macrostachyus, Acacia nilotica, Ekebergiacapensis, Clematis hirsute and Adenia gummifera. The 8 plant extractswere selected from 23 total plant extracts which had been previouslyassayed against E. coli and S. aureus. As shown in Table 2, CD4 countsincreased of subjects by up to 100 per month, but none of the subjectstested HIV negative within the three-month period.

TABLE 2 CD_(4/uL) per month Subject ID Month 1 Month 2 Month 3 Month 4Month 5  1b 118 150 399 420 —  2b 100 250 420 460 —  3b 04 93 190 320 — 4b 667 550 815 830 —  5b 160 120 480 620 —  6b 210 190 520 510 —  7b420 500 780 780 —  8b 128 108 310 304 —  9b 110 150 380 348 — 10b 380460 716 716 — 11b 300 410 390 560 — 12b 100 120 310 318 — 13b 250 180340 420 — 14b 80 70 260 380 — 15b 140 110 300 420 — 16b 250 180 290 360— 17b 300 380 460 580 — 18b 280 290 290 410 — 19b 118 190 170 320 — 20b160 160 220 299 360

Example 4

In another experiment, 21 HIV-positive subjects were treated with aherbal composition consisting of the 14 herbal ingredients identified inTable 1. Subjects were administered a composition prepared by dissolvingapproximately 1 tbsp. (or 15 ml) of the powdered ingredients (a mixtureprepared the 14 plants listed in Table 1) in approximately 8 ozs. (250ml) of hot water. The supernatant liquid was then ingested by thesubject.

The subjects were divided into two groups: the first group having 10subjects (subject ID Nos. 1-10) and the second group having 16 subjects(Subject ID Nos. 11-26). In the first group, each the 14 plants waspresent in the composition in equal weight ratios. In the second group,the concentrations of Dovyalis abyssinica and Clutia robusta wereapproximately half of the other 12 ingredients as disclosed.

As shown in Table 3, CD4 counts for each subject were measured on amonthly basis. The CD4 counts of the test subjects treated with the 14ingredient herbal composition increased by up to 100 per month. Sixsubjects tested HIV-negative after four months of treatment. Twosubjects tested HIV-negative after two months of treatment.

TABLE 3 Subject CD_(4/uL) per month ID Month 1 Month 2 Month 3 Month 4 1420 450 570 HIV negative 2 320 390 480 520 3 100 115 250 — 4 80 150 310— 5 340 370 480 560 6 120 180 299 — 7 118 350 360 HIV negative 8 125 105225 — 9 300 200 400 HIV negative 10 280 399 410 HIV negative 11 400 500520 HIV negative 12 250 250 310 — 13 250 460 600 — 14 400 520 780 — 15250 330 480 HIV negative 16 667 550 815 830 17 150 250 380 — 18 620 640660 — 19 310 400 480 — 20 243 245 280 — 21 180 216 434 — 22 280 390 — —23 360 420 — — 24 190 280 — — 25 630 720; — — HIV negative 26 N/A; N/A;HIV positive HIV negative

By comparison with the results achieved with the present invention, in astudy conducted on subjects on HAART in Moi University Teaching andAcademic Model for Prevention and Treatment of HIV (AMPATH), the CD4count increases were gradual, generally taking several years to reachabove 500. The subjects were treated with conventional antiretroviral(ARV) therapy, consisting of twice daily dosing of Stavudine, Lamivudineand Nevirapine (d4T-3TC-NVP). Other ARV regimes include treatment withcombinations consisting of ZDV-3TC-NVP, d4T-3TC-EFV and ZDV-3TC-EFV(wherein ZDV is Zidovudine and EFV is Efavirenz). Treatment guidelinesare provided in the publication “Integrated Management of Adolescent andAdult Illness,” published in January 2004 by the World HealthOrganization. ARV therapy subjects rarely reverse their seroconversionstatus, and among those listed in Table 4, none did so.

TABLE 4 Comparative Results of CD4 Count Increases in Subjects UnderConventional ARV Therapy. 6 Months 1 Year 1½ Years 2 Years 2½ Years 3Years 1. 247 207 264 197 138 367 2. 315 327 150 260 — — 3. 268 199 195360 — — 4. 99 163 — — — — 5. 265 40  36 247 332 397 6. 138 311 584 578 —— 7. 37 298 — — — — 8. 201 261 — — — — 9. 21 52  74 309 — — 10. 2 156 —— — — 11. 43 200 — — — — 12. 169 295 — — — — 13. 75 144 179 — — —

All patents and other references cited in the specification areindicative of the level of skill of those skilled in the art to whichthe invention pertains, and are incorporated by reference in theirentireties, including any tables and figures, to the same extent as ifeach reference had been incorporated by reference in its entiretyindividually.

One skilled in the art would readily appreciate that the presentinvention is well adapted to obtain the ends and advantages mentioned,as well as those inherent therein. The methods, variances, andcompositions described herein as presently representative of preferredembodiments are exemplary and are not intended as limitations on thescope of the invention. Changes therein and other uses will occur tothose skilled in the art, which are encompassed within the spirit of theinvention, are defined by the scope of the claims.

Definitions provided herein are not intended to be limiting from themeaning commonly understood by one of skill in the art unless indicatedotherwise.

The inventions illustratively described herein may suitably be practicedin the absence of any element or elements, limitation or limitations,not specifically disclosed herein. Thus, for example, the terms“comprising”, “including,” containing”, etc. shall be read expansivelyand without limitation. Additionally, the terms and expressions employedherein have been used as terms of description and not of limitation, andthere is no intention in the use of such terms and expressions ofexcluding any equivalents of the features shown and described orportions thereof, but it is recognized that various modifications arepossible within the scope of the invention claimed. Thus, it should beunderstood that although the present invention has been specificallydisclosed by preferred embodiments and optional features, modificationand variation of the inventions embodied therein herein disclosed may beresorted to by those skilled in the art, and that such modifications andvariations are considered to be within the scope of this invention.

The invention has been described broadly and generically herein. Each ofthe narrower species and subgeneric groupings falling within the genericdisclosure also form part of the invention. This includes the genericdescription of the invention with a proviso or negative limitationremoving any subject matter from the genus, regardless of whether or notthe excised material is specifically recited herein.

The plant parts described in the specification are those in which in theexperience of the inventors, the highest concentration of beneficialingredients are to be found. However, it will be apparent to thoseskilled in the art that the same or other beneficial compounds may befound in other parts of the recited plants not specifically recited inparagraph [0022] above and elsewhere in this application, and thattherefore, any composition comprised of any part or parts of the recitedplants which includes Dovyalis abyssinica and Clutia robusta is withinthe scope of the invention.

Other embodiments are within the following claims. In addition, wherefeatures or aspects of the invention are described in terms of Markushgroups, those skilled in the art will recognize that the invention isalso thereby described in terms of any individual member or subgroup ofmembers of the Markush group.

1. A process for preparing an herbal composition mixing dried root ofClutia robusta and dried root of Dovyalis abyssinica to form a mixture,boiling the mixture in water to form a water extract, and filtering thewater extract to obtain a filtrate, thereby preparing the herbalcomposition.
 2. A process for extracting alkaloid compounds from anherbal mixture comprising: grinding and mixing Dovyalis abyssinica andClutia robusta to form an herbal mixture; extracting said herbal mixturewith a non-polar solvent; adding base to obtain an basic solution andheating said mixture; adding acid to obtain an acidic solution andheating said mixture; extracting said acidic solution with a non-polarsolvent; and precipitating and collecting the alkaloid compounds.
 3. Themethod of claim 2 wherein the base is NaOH(_(aq)).
 4. The method ofclaim 2 wherein the acid is HCl(_(aq)).
 5. The method of claim 2 whereinthe acidic solution is heated at a simmer for about 3 hours.
 6. Themethod of claim 2 wherein the non-polar solvent is ether.
 7. The methodof claim 2 wherein the non-polar solvent is xylene.
 8. The method ofclaim 2 wherein the pH of the basic solution is about
 8. 9. The methodof claim 2 wherein the pH of the acidic solution is about
 3. 10. Themethod of claim 2 further comprising decanting the acidic solution toprovide an acidic extract and a residue, adding acid to the residue andheating the acid and residue at a simmer for about four hours.
 11. Themethod of claim 2 wherein the non-polar solvent added is about 20% byvolume.
 12. The method of claim 2 wherein the alkaloid precipitation isperformed at a pH of about
 9. 13. The process of claim 2, wherein in thegrinding and mixing step dried stem bark of Prunus Africana, dried stembark of Croton macrostachyus, dried stem bark of Acacia nilotica, ordried root of Periploca linear ifolia are included to form the herbalmixture.
 14. The process of claim 2, wherein in the grinding and mixingstep dried stem bark of Prunus Africana, dried stem bark of Crotonmacrostachyus, dried stem bark of Acacia nilotica, and dried root ofPeriploca linear ifolia are included to form the herbal mixture.
 15. Themethod of claim 2, wherein dried root of Dovyalis abyssinica and driedroot of Clutia robusta are used and wherein dried root of Dovyalisabyssinica and dried root of Clutia robusta are used in a weight ratioof 1:1.
 16. The method of claim 14, wherein the dried root of Dovyalisabyssinica, the dried root of Clutia robusta, the dried stem bark ofPrunus africana, the dried stem bark of Croton macrostachyus, the driedstem bark of Acacia nilotica, the dried root of Rhamnus prinoides, thedried root of Adenia gummifera, the dried root of Asparagus africanus,the dried stem bark of Anthocleista grandiflora, the dried whole plantof Plantago palmata, the dried root of Clematis hirsuta, said dried stembark of Ekebergia capensis, the dried stem bark of Bersama abyssinicaand the dried root of Periploca linearifolia are in a weight ratio of2:2:2:2:2:2:1:2:2:1:2:2:2:2.